Methods of nucleic acid amplification

ABSTRACT

The present invention provides a method of simultaneously amplifying a plurality of target sequences within sample nucleic acid which comprises: (1) contacting said sample nucleic acid with one or more primer pairs under conditions which allow hybridisation of the primers to the sample nucleic acid, each primer having a bipartite structure A-B wherein part A is specific for a particular target sequence within the sample nucleic acid and part B is a constant sequence which is common to all primers or is common amongst all forward primers with a different sequence common amongst all reverse primers; (b) performing first amplification reaction; (c) degrading the bipartite primers or separating them from the amplification products of the first amplification reaction; (d) contacting the amplification products from the first amplification reaction with primers which comprise part B of the bipartite primers or a nucleotide sequence which is substantially identical to part B, under conditions which allow hybridisation of the primers to the amplification products; and (e) performing a second amplification reaction and kits for use in such methods.

The present invention relates to methods of nucleic acid amplification,in particular to methods that employ the polymerase chain reaction(PCR).

DNA amplification techniques, and in particular the polymerase chainreaction (PCR) have become key diagnostic tools. Theoretically, a singletarget molecule can be detected in a background of 10¹⁰ to 10¹²non-target molecules. Recently, technology has been developed thatallows nucleic acid quantification by monitoring the PCR amplificationreaction in real-time (Orlando, C. P. Pinzani, and M. Pazzagli. 1998.Clin Chem Lab Med. 36(5):255-69.). There have also been efforts in theamplification of several targets simultaneously (multiplex PCR)(Elnifro, E. M., A. M. Ashshi, R. J. Cooper, and P. E. Klapper. 2000.Clin Microbiol Rev. 13(4):559-70.). This, however, is very complicatedsince several different primer pairs have to be optimisedsimultaneously.

While there is a demand in many diagnostic and other fields formultiplex PCR, the optimisation of multiplex PCR poses several problems,including poor sensitivity or specificity and/or preferentialamplification of certain targets. Primers with better than averagepriming efficiency will produce more of their product and potentiallyuse up the available triphosphates in the reaction mixture beforeamplicons relying on other less efficient primers reach detectablelevels.

In addition, the presence of more than one primer pair in the multiplexPCR reaction increases the chance of obtaining spurious amplificationproducts, primarily through the formation of primer dimers. Thesenon-specific products may be amplified more efficiently than the desiredtarget, consuming reaction components and producing impaired rates ofannealing and extension. The optimisation of multiplex PCR should aim tominimise or reduce such non-specific interactions.

Most diagnostic assays require detection and quantification of severaldifferent targets simultaneously. The methodological limitations, are inmany cases the reasons for developing simplex assays, or assaysincluding only a few targets. This is for instance the case with thecurrent tests for genetically modified organisms (GMOs, essentiallyplant material) in foods. Currently, about 50 GMO constructs areapproved in for commercial use in USA. In Europe, approved GMO foodsrequire labelling if more than 1% of any ingredient originates from aGMO. Considering the large numbers of GMOs expected in the future,multiplex quantitative measurements are required to determine whetherthe foods contain approved or unapproved GMO constructs, and whether theamount of GMO in the ingredients is above or below 1%.

Thus, while multiplex PCR is a very useful technique in theory, thepractical problems of simultaneously performing multiple reactions areholding back its use. Recently, a technique has been proposed (Shuber,A. P., V. J. Grondin, and K. W. Klinger. 1995. Genome Res. 5(5):488-93.)which seeks to reduce the impact of the different amplificationefficiencies of different primers. Such methods involve the performanceof two distinct PCR reactions, with different amplification primers usedin each reaction. The primers used in the first reaction are bipartite,each containing a region which is specific for a particular targetsequence within the nucleic acid sample to be analysed and a universalregion at the 5′ end.

Amplification cycles are performed to generate a population of ampliconsfrom each target sequence. The region which is specific for a giventarget sequence hybridizes to the sample nucleic acid so that normalpolymerase controlled extension can occur. The universal region does nothybridise with the original template nucleic acid but as products fromearlier cycles are used as templates, this constant segment and regionscomplementary to it are incorporated into the amplicons. This helps tonormalize the hybridisation kinetics across the different targetsequences being simultaneously amplified, preventing individual targetsequences being significantly over or under represented at the end ofthe reaction.

Then a second amplification reaction is performed using as primersoligonucleotides which comprise or consists of the universal region fromthe first amplification reaction. The different target regions are thusamplified using the same primers and the ratio of the number of statingmolecules to end product amplicons should therefore be constant.

Such a method is described, for example in WO 99/58721 which isincorporated herein by reference. This publication particularlyaddresses the problems of amplifying and detecting many different targetsequences in a single reaction and success is attributed to acombination of factors, including the small size of the amplificationtargets, optimization of amplification conditions and the presence ofthe constant (universal) sequence at the 5′-end of the primers.

However, in practice the methods described in WO 99/58721 and in J. Med.Genet 2000: 37 272-280, do not provide quantitative results in amultiplex PCR system. There are many scenarios where as well as testinga sample for the presence of a number of different nucleic acidsequences of interest (multiplex), it is desirable to determine thelevel of each sequence in a sample, i.e. to obtain quantitative results.Of particular interest is the need for food producers and food controlauthorities to test whether foods and food ingredients containgenetically modified plants. Already about 50 different geneticallymodified plants have been approved in the USA and it would clearly bevery costly and time consuming to analyse a food sample for specificgenetically modified plants (gmps) in a series of separate reactions. Asthe number of gmps increases and their use become more widespread itwill be desirable to use multiplex assays to detect signature geneticelements used in gmps in a single reaction. It is also desirable to haveinformation about whether the specific group is present in the food onlyin trace amounts or whether the amount is above or below a certainlimit. At present there are no methods available which reliably providethis quantitative information in a multiplex environment.

A method has now been developed which addresses these problems and hasbeen shown to provide quantitative multiplex PCR in the context ofdetecting gmps and which also has general applicability to assays wherequantitative results of multiplex PCR are required. The method is basedon the two step PCR described above but it has surprisingly been foundthat removal of the primers from the first amplification reactionensures that the second amplification reaction, and thus the method as awhole, retains its quantitative character. According to this methodtherefore, the second amplification reaction is performed in the absenceof the primers from the first amplification reaction.

Thus, according to one aspect, the present invention provides a methodof simultaneously amplifying a plurality of target sequences withinsample nucleic acid which comprises:

-   -   (a) contacting said sample nucleic acid with one or more primer        pairs under conditions which allow hybridisation of the primers        to the sample nucleic acid, each primer having a bipartite        structure A-B wherein part A is specific for a particular target        sequence within the sample nucleic acid and part B is a constant        sequence which is common to all primers or is common amongst all        forward primers with a different sequence common amongst all        reverse primers;    -   (b) performing a first amplification reaction;    -   (c) degrading the bipartite primers or separating them from the        amplification products of the first amplification reaction;    -   (d) contacting the amplification products from the first        amplification reaction with primers which comprise part B of the        bipartite primers or a nucleotide sequence which is        substantially identical to part B, under conditions which allow        hybridisation of the primers to the amplification products; and    -   (e) performing a second amplification reaction.

The primers used in the second amplification reaction (step (d)) willpreferably be identical or substantially identical to part B of thebipartite primers used in the first amplification reaction and willtypically not comprise part A or a functional part or equivalentthereof. The term ‘substantially identical’ will be understood withfunctional considerations in mind, i.e. the ability to hybridiseefficiently to the amplification products of the first amplificationreaction. Typically this will mean no more than 5 nucleotide additions,deletions or substitutions, preferably no more than 3. These primers‘comprise part B of the bipartite primers (or a nucleotide sequencewhich is substantially identical to part B)’, i.e. the nucleotidesequence of these primers comprises the same sequence as part B of thebipartite primers used in the first amplification reaction (or anucleotide sequence which is substantially identical to part B).

In a preferred embodiment, the constant region B of the bipartiteprimers is common between both forward and reverse primers and thus onlya single primer species is required in the second amplificationreaction. In an alternative embodiment, it may be desirable to havedifferent forward and reverse primers, with one of the primer specieslabelled for subsequent detection. Whether the constant region (B) iscommon to all primers or only amongst the forward or reverse primers, itis found at the 5′end of both the forward and reverse primers; thevariable section (A) which is designed to hybridise to a sequence in thesample nucleic acid is found at the 3′end of the bipartite primers. Thusthe abbreviation ‘A-B’ does not imply a relative position within themolecule for the two regions in terms of the 3′ and 5′ ends. Theconstant region (B) is typically 10-40 nucleotides in length, preferably12-25 nucleotides in length.

The region B will either be substantially the same in all bipartiteprimers or substantially the same amongst the forward primers with asecond region B′ which is different to B but is substantially the sameamongst all the reverse primers. Preferably B (or B′) will be exactlythe same in all bipartite primers or at least in all forward or allreverse primers but it will be understood that a small number ofnucleotide variations between sequences will not significantly affectthe method. The term ‘common’ should be interpreted with this in mind.The purpose of these constant regions is to even out differences inpriming efficiency and to provide highly efficient hybridisation andpriming with the primers used in the second amplification reaction.Therefore between B sequences which are substantially the same therewill preferably be variation at no more than 3 nucleotide positions.

Preferably the constant region(s) B is chosen so that it does nothybridise with the sample nucleic acid, or at least does not hybridiseefficiently therewith. Thus a randomly chosen sequence may beconstructed according to the well known rules for primer design.

Part A of the bipartite primers is specific for particular targetsequences in that they are designed to hybridise to a region of nucleicacid which flanks the target sequence which it is desired to amplify.According to the normal conventions of the PCR, the A sequences will bein pairs, each pair consisting of a forward primer and a reverse primerwhich hybridise to regions upstream and downstream of a nucleotidesequence of interest. The bipartite primers will therefore be formedinto pairs of forward and reverse primers by the nature of their Asequence. In certain cases, part A may be substantilly identical in theforward and the reverse primer, for example when the desired targetsequence is flanked by inverted repeats as is often the case with mobileelements such as transposons.

The primers may have the form A^(F1)-B, A^(R1)-B, A_(R1)-B, A^(R2)-Betc. where ‘A^(F1)’ indicates a forward primer sequence which hybridisesto a flanking region of a first target sequence and ‘A^(R1)’ a reverseprimer sequence which hybridises to the other flanking region of thefirst target sequence. As mentioned above, the common regions B may bedifferent in forward and reverse primers, thus having the form A^(F1)-B,A^(R1)-B′, A^(F2)-B, A^(R2)-B and so on.

The part A regions which hybridise to specific-regions in the samplenucleic acid amplification are selected by methods well known in thefield of nucleic acid amplification. In order to select a pair of Asequences for amplifying a target region, the sequence of and adjacentto the target sequence must be known (or at least approximately known).Short stretch sequences at either end of the target sequence are thenselected and the primers designed for hybridisation to these regions.

Typically only a few cycles will be performed in the first amplificationreaction, e.g. less than 25, preferably less than 15, more preferablyless than 10, to avoid potential artefacts in the multiplexamplification and to ensure that none of the targets reach saturationlevels. Preferably this first amplification reaction is carried outusing standard PCR reagents and conditions and suitable parameters forthe cycles are described in the examples and are generally well known inthe art. The ‘first’ and ‘second’ amplification reactions thereforerefer to two sets of amplification cycles, each defined by the primersinvolved.

To increase amplification efficiency for a given target sequence, theprimer concentrations for that target may be increased for the firstamplification reaction.

The bipartite primers are then separated from the amplification productsof the first amplification reaction before the second amplificationreaction takes place. By ‘separation’ is meant the separation of thebipartite primers and the amplification products into two distinctpools, not the dissociation of primer and template which occurs as anintegral part of all standard PCR reactions. This may be achieved byremoving the bipartite primers, conveniently this is done by breakingdown the bipartite primers e.g. by exonuclease degradation.Alternatively, the bipartite primers could possess a non-standardmodification, e.g. contain uracil instead of thymine, and couldtherefore be degraded by a DNA-modifying enzyme such as uracil-DNAglycosylase. This enzyme removes uracil from the sugar backbone whichleads, on heat treatment, to a strand break. This enzyme removes uracilfrom the sugar backbone which leads, on heat treatment, to a strandbreak. The use of bipartite primers which contain uracil only in the Apart would allow the selective degradation of only this part, leavingpart B intact, which could then participate in the second amplificationreaction.

Thus, reference above to ‘degradation’ of the bipartite primers includesboth full or partial degration and so a molecule which has beenpartially broken down can be considered to be degraded. It is importantthat the A parts of the bipartite primers are no longer available totake part in hybridisation reactions during the second amplificationreaction. A ‘DNA-modifying enzyme’ is therefore able to inactivate thebipartite primers or at least part A thereof.

Alternatively the amplification products may be isolated from the restof the initial reaction mixture which contains the bipartite primers.The products of the first amplification reaction are thus purifiedbefore being used as templates for the second amplification reaction.Purification is conveniently achieved by capturing the amplificationproducts on a solid support e.g. by using a standard PCR productpurification kit or through attaching a binding moiety to theamplification products and providing a binding partner for said bindingmoiety on the solid support. The binding moiety may be attached to aprobe which in turn hybridises to the amplification product. Suitablebinding moieties are well known in the art and include,streptavidin/biotin, antigen/antibody interactions, lectin bindingsystems or probes covalently bound to a solid support etc. Suitablesolid supports are also well known and widely available, preferably thesupport is magnetic and particulate for ease of manipulation.

Key to step (c) is the fact that all or most, i.e. at least 70%,preferably at least 80%, more preferably at least 90% of the bipartiteprimers are degraded or separated from the amplification products beforethe second amplification reaction takes place.

The second amplification reaction uses either a single primer species ora single forward primer species and a single reverse primer species. Ifappropriate, these may be present in the reaction mix from the start,i.e. during the first amplification reaction, or be generated throughthe modification or partial degradation of the bipartite primers, forexample as described above where uracil replaces thymine in part or allof the bipartite primers. In other embodiments of the invention, e.g.where an exonuclease is used to degrade the primers or the amplificationproducts are separated from the bipartite primers through the use of alabelled probe, the primers used in the second amplification reactionwill not be present in the initial reaction mix. Step (d) of the methoddefined above therefore encompasses all of these variants.

It may be desirable to perform all the method steps (i.e. both the firstand second amplification reactions) in one reaction vessel anddegradation of the bipartite primers may conveniently allow this.

The advantages of the methods of the invention are twofold. Onelimitation of multiplex PCR is the different amplification efficienciesof the different amplicons when specific primer sets are used. This willlead to a situation where some of the amplicons present are amplifiedwhereas others are not. In addition, using many different primer pairsin one reaction inevitably leads to a large number of side reactions dueto primers interacting with each other. These side reactions perturb thePCR. The use of a constant part B in the first step primers incombination with the removal of those primers eliminates these problems.Secondly, the amplification of all targets with the same primer orprimer pair leads to a constant ratio of the different targets in themultiplex PCR before and after amplification, in the same way as incompetitive PCR. By effectively removing the bipartite primers after thefirst PCR step, these do not interfere with the ratios of the differentamplicons during the second PCR step. This removal is what makes thesystem maintain its quantitative nature.

‘Amplification’ refers to a process for using polymerase and a pair ofprimers for increasing the amount of a particular nucleic acid sequence,a target sequence, relative to the amount of that sequence initiallypresent in the sample nucleic acid. Amplification may conveniently beachieved by the in vitro methods of PCR (including reverse transcriptasePCR (RT-PCR)) or ligase chain reaction or others as well as NASBA(nucleic acid sequence based amplifications) approaches.

A ‘target sequence’ is a sequence that lies between the hybridisationregions of a pair of primers (and may in addition include the primersequences themselves) and can be amplified by them. The number ofdifferent target sequences within the sample which may be amplified willdepend on the nature and requirements of the assay. Typically there willbe more than 4, e.g. 8 or more even 12 or 20 or more different targetsequences amplified in one multiplex reaction.

In the context of assaying for the presence of GMOs, the targetsequences may fall into one of a number of categories. The targetsequence may fall entirely within a gene of interest and the ampicillinPCR in the multiplex system described in the present Examples is anexample of this. The ampicillin resistance gene is included in pUC18which is used in the generation of Bt176 corn (Maximizer Corn). Apositive PCR result shows the presence of the gene but does notdetermine the origin of the DNA and therefore the amp signal couldoriginate from Bt176 DNA but could also originate from a bacterialcontamination of the plant.

A gene of interest is typically part of a construct of interest and apromoter often used in such constructs is the 35S promoter from theCauliflower mosaic virus (CaMV). One of the PCRs in the multiplex PCRdescribed in the present examples detects this promoter and thus targetsequences may be in regulatory regions. Although again, a positiveresult may indicate that the plant has been infected with Cauliflowermosaic virus. The nos reaction of the present examples detects adifferent regulatory region used in these constructs, the NOSterminator.

A more specific approach is to design a primer pair overlapping ajunction region between a promoter or terminator (a regulatory region)and a gene of interest. These DNAs do not occur naturally in nature andthus a PCR signal would be a very strong indication of the presence ofGMOs. In the present examples such an overlap is detected in themultiplex PCR system for Bt176 and Bt11 (Methods for the specificdetection of Bt176 corn and Bt11 corn are described in Hurst, C. D. etal. (1999) European Food Research and Technology Vol. 5, 579-586 andZimmermann, A. et al. (2000) Lebensmittel-Wissenschaft & Technologie,33, 210-216 respectively). In the Bt176 PCR a fragment overlapping thejunction between the pepC promoter (phosphoenol pyruvate carboxylasepromoter from maize) and the cry gene (a synthetic gene from Bacillusthuringiensis which confers insect resistance) is targetted. In Bt11 thePCR overlaps the junction between the 35S promoter and an enhancer DNAfragment from the alcohol dehydrogenase gene from maize. In a preferredembodiment of the present invention, one or more of the target sequencesspans a non-naturally occurring nucleic acid sequence, e.g. a sequencecomprising regions which are not naturally found in juxtaposition.

However, even this approach could conceivably cause problems if acompany used the same construct, e.g. a specificpromoter-enhancer-gene-terminator in several different plants (be it thesame species or not, but different transformation events). One of thesetransformations (GMOs) may be approved by the relevant regulatory bodywhile others are not but the PCR would not be able to discriminatebetween the approved GMO and the non-approved GMO(s). When a plant istransformed, DNA integrates randomly at different sites for eachtransformation event. Thus a way of overcoming the problems discussedabove would be to determine the plant DNA sequence which flanks theinserted DNA, and then construct a primer pair which overlaps thisjunction (which can be called an ‘event specific region’).

Thus in a preferred embodiment of the methods of the present inventionone or more of the target sequences is for an event specific region,i.e. spans a region which comprises both host plant species DNA andinserted DNA from the genetically engineered construct.

The Mon810 PCR of the present examples is an example of such an eventspecific region (Zimmermann et al. (1998)) Food Science and Tech. 31,664-667 have designed a nested PCR system for the detection of Maisgardcorn (Mon810 corn) as the amplified sequence lies in the overlap betweenintegrated DNA and the plant's endogenous DNA.

The sample nucleic acid may be isolated or may exist as part of a mixedsample which includes other cellular components from the biologicalsource from which it was obtained. Methods of isolating nucleic acidfrom a biological sample are well known in the art. Any biologicalsample containing nucleic acid is a suitable source of nucleic acid andthus the sample may be derived from animals, plants, insects, bacteria,yeast, viruses or other organisms. Particularly preferred sources ofsample nucleic acid for amplification according to the present inventionare plants or food products which contain or are suspected of containinggenetically modified material. The ‘sample nucleic acid’ may be derivedfrom one or more biological samples. In the context of plants andfoodstuffs for example, a single plant may provide the sample nucleicacid or it may be derived from a number of plants of the same or evendifferent species.

By ‘nucleic acid’ is meant DNA (including cDNA) or RNA. The nucleic acidmay be naturally occuring or synthesised by chemical or recombinanttechniques.

The above amplification method is then generally followed by a detectionstep and suitable detection methods for multiplex PCR are known in theart and discussed, for example, in WO 99/58721. When performing amultiplex reaction it is necessary to differentiate between theamplification products from different loci. This could be done on thebasis of size discrimination, e.g. on gels but requires theamplification products to be of different sizes, e.g. 100 bp, 200 bpetc. The reaction products could be differentially labelled, i.e.different tags are attached to primers for different loci, however sucha technique is limited by the number of different commercially availabletags (e.g. fluorescent molecules).

Thus in a preferred embodiment probes specific to the differentnucleotide sequences of interest which have been amplified areenzymatically labelled at their 3′end and then the labelled probes arecaptured by hybridisation to complementary DNA on a solid support e.g.nylon filters, glass slides, chips etc. Such methods are described inthe Examples and in WO 99/50448.

These probes to the different target regions may be labelled at the5′-end with a fluorescent group other than the one used in the 3′-endlabelling reaction. During fluorescent scanning it would then bepossible to calculate immediately the percentage of molecules labelledduring the labelling reaction.

As discussed above, the methods claimed herein are quantitative innature. The signal strengths for identified target sequences can becompared to known standards to calculate the concentration (e.g. copynumber) of that target sequence in the sample. As described in theExamples, a known concentration of a control sequence (IPC) mayconveniently be added to the sample to adjust for fluctuations inamplification efficiency from one sample reaction mix to another; theuse of such an internal control determines the absolute amount ofnucleic acid in the sample and is a preferred embodiment of the presentinvention. In a further preferred embodiment, also described in theExamples, a species specific target sequence is amplified and thisreference gene enables the relative amounts of nucleic acidconstructs/sequences of interest (e.g. a target GM construct) ascompared to the material from said species to be determined.

Thus, the invention provides data for a given target sequence which canbe quantified against a known reference for that target sequence. Targetsequences can be detected qualitatively and quantitatively according tothe methods of the invention and the results from different experimentscompared because quantifiable information is obtained.

In a further aspect, the present invention provides a kit for use in amethod of nucleic acid amplification, typically any method as describedabove, which comprises:

-   -   (a) a plurality of bipartite primer pairs of form A-B as defined        above;    -   (b) means for degrading the bipartite primers or for separating        them from the amplification products of a first amplification        reaction; and optionally    -   (c) primers which comprise part B of the bipartite primers of        component (a) or a nucleotide sequence which is substantially        identical to part B of said primers.

Means (b) may conveniently include exonucleases which degrade theprimers, standard PCR-product purification kits or probes that capturethe amplification products on a solid support. Where part A but not partB of the bipartite primers contains thymine, means (b) may convenientlycomprise an enzyme such as uracil DNA glycosylase which selectivelydegrades part A of the bipartite primers, generating primers for use ina further amplification reaction. In which case, a separate component(c) may not be required.

The invention will be further described in the following non-limitingexamples and with reference to the Figures in which:

FIG. 1 provides a schematic representation of the quantitative multiplexamplification method. (A): In the first PCR step, the targets areamplified with primers containing “heads” that are equal for all thetargets. (B): The “head”-containing primers are then removed byenzymatic digestion (left) or the amplified products are hybridized toan internal biotinylated capture probe and the complex is then purifiedthrough binding to biotinylated paramagnetic beads (right). These aretwo independent alternative purification strategies. (C): In the secondPCR step, a primer identical to the “head” sequence is used.

FIG. 2 provides a schematic illustration of the test format. The probescomplementary to the labelled test probes used in the enzymaticlabelling are spotted horizontally using a grid. During hybridisationthe grid is turned 90 degrees before application of hybridisationsolutions and labelled probes.

FIG. 3 shows multiplex detection of GMO corn samples. GM corn DNA wasanalysed either alone or in combinations. Line 1: detection of the cornreference DNA, line 2: Mon810 signals, Line 3: Bt11 signals, Line 4:Bt176 signals. The samples analysed are indicated under thecorresponding lanes (all analyses in duplicate), Lane 1,2: non GMOmaize, lane 3, 4: 0.4% Bt176, 0.7% Bt11 and 0.4% Mon810 DNA, lane 5, 6:1% Bt11 and 0.5% Bt176, lane 7, 8: 1% Mon810, lane 9,10: 1% Bt176, lane11, 12: 2% Bt11.

FIG. 4 shows quantitative chromogenic detection of Bt11 corn DNA usingthe multiplex assay. 2% Bt11 corn DNA was diluted in non GM corn DNA togive different concentratons of GM corn. The results show thequantitative response of the assay as the concentration of GM corn islowered. The first line shows the corn DNA reference signals, the secondrow shows the Bt11 signals. The signals were recorded on a Typhoonscanner, PE systems.

FIG. 5 shows eight-plex detection of GM maize. Eight specific primerpairs with “heads” were used in the first PCR step. The lines represent(from above): Bt 176, Bt11, Mon810, amp, Nos terminator, 35S promoter,Internal PCR control (IPC) and maize reference gene. Lanes 1 and 2: 2%Bt 176 maize DNA, lanes 3 and 4: 1% Bt11 maize DNA, lanes 5 and 6: 1 PC.

FIG. 6 shows quantitative multiplex PCR for detection of GM corn and thenecessity of removing primers after the 1. PCR step. Each line shows thedetection of a specific PCR product as indicated to the left. Each lane(a through 1) represent different samples. All samples (a-j) contained amixture of 0.7% Bt176 and 0.7% Bt11. In addition Mon 810 corn DNA wasadded to 2.0% (lanes a, b), 1.0% (lanes c, d), 0.5% (lanes e, f), 0.2%(lanes g, h) and 0.0% (lanes i, j). In addition all lanes (a-1)contained approx. 100 copies of an internal positive control (IPC) DNA.Amp: ampicillin resistance gene from the pUC18. Nos: Nos terminator,35S: Cauliflower mosaic virus promoter.

(A): PCR carried out in two steps: 1. PCR (10 cycles) using specificprimers with a common “head” sequence. Primers are then digested and the2. PCR (30 cycles) is carried out using the common head primer. (B) Sameas A, but the specific primers were not degraded before the 2.PCR step.Panel I: shows the fluorescence signals after hybridisation andscanning, panel II: shows the blot after binding of antibodies andenzymatic HRP colour reaction.

FIG. 7 illustrates the effect of omitting the 2. PCR step. Same as inFIG. 6A, except that the 1.PCR step using the specific primers with headsequence was extended to 40 cycles and the 2. PCR step was omitted.Panel A shows the fluorescence signals after hybridisation and scanning,panel B shows the blot after binding of antibodies and enzymatic HRPcolour reaction.

FIG. 8 illustrates the effect of diluting the template DNA. A referencemixture of 0.7% Bt176, 0.7% Bt11 and 0.7% Mon810 at different dilutionswas used as templates in the PCR. Panel I shows the fluorescence signalsafter hybridisation and scanning, panel II shows the blot after bindingof antibodies and enzymatic HRP colour reaction. Lanes 1, 2: undilutedDNA template, lanes 3, 4: 1/4 dilution, lanes 5, 6: 1/16 dilution, lane7, 8: 1/64 dilution, lanes 9, 10: 1/256 dilution, lanes 11, 12: notemplate added.

FIG. 9 shows quantitative 8-plex detection of Mon810 DNA alone (A) ortogether with 2% Bt11 DNA (B). Panel I shows the fluorescence signalsafter hybridisation and scanning, panel II shows the blot after bindingof antibodies and enzymatic HRP colour reaction. Lanes 01, 02: Areference mixture of 0.7% Bt176, 0.7% Bt11 and 0.7% Mon810. Lane 1a, 1b:5% Mon810, lanes: 2, 3: 2% Mon810, lanes 4, 5: 1.0% Mon810, lanes 6, 7:0.5% Mon810, lanes 8, 9: 0.1% Mon810, lanes 10, 11: 0% Mon810, lanes 12,13: IPC (date 020901).

FIG. 10 shows the relationship between amount of Mon810 maize in asample and the signal strength. The Mon810 fluorescence signals in FIG.9 panel I, were quantified using Imagemaker program and plotted againstthe given concentration of the samples.

FIG. 11 illustrates quantitative 8-plex detection of Mon810 DNA alone(A) or together with 2% Bt11 DNA (B). Repetition of example 6 (FIG. 9).Panel I shows the fluorescence signals after hybridisation and scanning,panel II shows the blot after binding of antibodies and enzymatic HRPcolour reaction. Lanes 01, 02: A reference mixture of 0.7% Bt176, 0.7%Bt11 and 0.7% Mon810. Lane 1a, 1b: 5% Mon810, lanes: 2, 3: 2% Mon810,lanes 4, 5: 1.0% Mon810, lanes 6, 7: 0.5% Mon810, lanes 8, 9: 0.1%Mon810, lanes 10, 11: 0% Mon810, lanes 12, 13: IPC (date 130901).

FIG. 12 shows the relationship between amount of Mon810 maize in asample and the signal strength. The Mon810 fluorescence signals from theexperiment in FIG. 11 were quantified using Imagemaker program andplotted against the given concentration of the samples. The average of 2parallels are shown.

FIG. 13 illustrates quantitative 8-plex detection of Bt176 DNA alone (A)or together with 1% Mon810 DNA (B). Panel I shows the fluorescencesignals after hybridisation and scanning, panel II shows the blot afterbinding of antibodies and enzymatic HRP colour reaction. Lanes 1, 2: Areference mixture of 0.7% Bt176, 0.7% Bt11 and 0.7% Mon810. Lane 3, 4:2% Bt176, lanes: 5, 6: 1% Bt176, lanes 7, 8: 0.5% Bt176, lanes 9, 10:0.2% Bt176, lanes 11, 12: 0.1% Bt176, lanes 13, 14: 0% Bt176, lanes 15,16: IPC (date 060901).

FIG. 14 shows the relationship between amount of Bt176 maize in a sampleand the fluorescence signal strength. The Bt176 fluorescence signalsfrom the experiment in FIG. 13 were quantified using Imagemaker programand plotted against the given concentration of the samples. The averageof 2 parallels are shown.

FIG. 15 Twelve-plex system for detection of seven different GM maizeevents. HRP enhanced chromogenic signals are shown. Samples 1, 2: amixture of 0.7% of each of Mon810, Bt11 and Bt176 and 1% of each of T25,GA21, CBH351 and DBT418, 3, 4: non-GM maize, 5, 6: 2.0% CBH351, 7, 8:0.5% CBH351, 9, 10: 2% DBT418, 11, 12: 0.5% DBT418, 13, 14: 2% GA21, 15,16: 0.5% GA21, 17, 18: 2% T25, 19, 20 0.5% T25. Amplicons are asdescribed for the eight plex PCR in FIG. 9. with additions of ampliconsfor CBH351, DBT418, GA21 and T25 given in Table 1.

FIG. 16 Twelve-plex system for detection of seven different GM maizeevents. Quantifications of the fluorogenic signals for CBH351, DBT418,GA21 and T25 from the experiment shown in FIG. 15. (□) Signals obtainedfrom samples 1 and 2 of FIG. 15.

Example 17 Screening of commercial samples using the 12-plex PCR system.The results after chromogenic enhancement is shown. Samples 01, 02:Reference mix containing 0.7% of each of Bt176, Bt11 and Mon810, 03, 04:Reference mix containing 2% of each of CBH351, DBT418, GA21 and T25.1-19: Food and feed samples. All samples contained approx. 100 copies ofIPC (internal positive control).

FIG. 18 Quantification of food and feed samples from USA with regards toMon810 using the eight-plex PCR system. Samples are as indicated in theFigure. US maize sample no. 4 is an additional maize meal referencecontaining 1% Mon810. All samples were analysed in duplicate.

Example 19 Quantification of food and feed samples from USA with regardsto Bt11 and Bt 176 using the eight-plex PCR system. Samples are asindicated in the Figure. All samples were analysed in duplicate.

FIG. 20 Comparisons of standard deviations for multiplex PCR and5′nuclease PCR on food and feed samples from USA per. Yellow column:averaged standard deviation for 5′ nuclease PCR in a European ringtrialfor determination of Bt176. The ringrial included six maize mealsanalysed by nine different laboratories.

EXAMPLES

Materials and Methods

Template and DNA Purification.

The method chosen exploits the use of DNA adsorption columns provided byQiagen in the DNeasy plant mini kit. Samples were homogenised whennecessary and purified as described by the manufacturer with thefollowing modifications.

The initial buffer volume was doubled and lysis was carried out for 30min at 65° C. using a shaking incubator. When eluting DNA bound to thecolumn, 50 μl of preheated buffer was used. In the repeated elution stepanother 50 μl buffer was added and the columns were spun at 13000 rpmfor 2 min.

The criteria used for assessing the quality of the DNA preparation wasthat no inhibition should be detected when samples were analysed withdifferent BioInside kits. This is easily seen on the internal PCRControl (IPC) provided by BioInside. Also the quality of DNA wasanalysed carrying out PCR on dilutions of a sample and calculating theamplification efficiency and quantifiable range of the PCR by plottingthe Ct values against the log DNA concentration and performing linearregression analysis. A large number of different food samples (>100)have been analysed giving good results with this DNA purificationmethod.

The maize reference gene used herein is the maize zein gene.

PCR amplification. Purified DNA was used in the amplification reactions.We used a two step PCR amplification approach (see FIG. 1 for aschematic representation). In the first step we used primers with both a5′-universal “head” and a gene specific region (see Table 1 below whichshows the specific regions of the bipartite probes, the biotin labelledisolation probe (as this may be used in place of degradation to separateprimers from amplification products) and the GM specific probes whichare labelled and then take part in DNA array hybridisation).

Primers with a “head” were then removed by enzymatic degradation or bytransfer of the PCR products to new tubes by capturing DNA ontoparamagnetic beads labelled with specific capture probes. In the secondPCR step a primer identical to the universal “head” region was used.

In the first PCR step we used 10 pmol of each of the primers, 1×Dynazyme DNA polymerase reaction buffer, 10 mM dNTP, and 2 μl DynaZymeDNA polymerase (2U μl) in a final volume of 50 μl. In some cases (forBt11 detection) the concentration of primers was increased. Theamplification protocol used was as follows (1.PCR step); 4 cycles usingthe parameters 95° C. for 30 s, 55° C. for 30 s, and 72° C. for 30 s,and then 6 cycles using the parameters 95° C. for 30 s and 72° C. for 30s. Twenty μl of the amplification product from PCR step 1 were treatedwith 2 μl Exonuclease I to degrade the residual single stranded primers,and 3 μl shrimp alkaline phosphatase to inactivate the nucleotides. Thereaction was incubated at 37° C. for 30 min, and then at 95° C. for 10min to inactivate the added enzymes.

Five μl of the exonuclease treated products were then used for thesecond PCR amplification step. 50 pmol of a universal primer identicalto the universal region (“head”) of the primers used in the first PCRreaction were added. The other components were the same as in the firstamplification. The amplification conditions used were: 95° C. for 15 sand 70° C. for 45 s for 40 cycles. During the course of the work somechanges and modifications in the PCR conditions were adopted. In theintroductory experiments the 2. PCR step was carried out under thefollowing conditions: 40 cycles of 95° C. for 15 s, 65° C. for 15 s and72° C. for 30 s. Later (pertaining to FIGS. 3, 4 and 5): the conditionswere changed to 95° C. for 15 s and 70° C. for 45 s for 40 cycles. Inlater experiments the same conditions were used but the number of cycleswere reduced to 30. In the final experiments the number of cycles in the1.PCR was reduced to 4, and the number of cycles in the 2. PCR stepagain increased to 40.

Sequence Specific Labelling.

After amplification with the “head” primer the amplification productswere treated with 2 μl exonuclase 1 and 2 μl shrimp alkaline phosphataseat 37° C. for 30 min, and then 95° C. for 10 min to inactivate theenzymes. The cyclic labelling conditions were as follows; 1×Thermosequenase reaction buffer, 10 pmol of each GM specific probe, 100pmol ddNTP (except ddCTP), 100 pmol Fluorescein-12-ddCTP, 16 UThermosequenase DNA polymerase, and 24 μl phosphatase and exonucleasetreated PCR product. The labelling was done using the followingparameters; 95° C. for 15 s, 60° C. for 1 min for 15 cycles, 95° C. for15 s, 55° C. for 1 min for 15 cycles, and finally 95° C. for 15 s, 50°C. for 1 min for 15 cycles.

DNA Array Hybridisation.

The format of the assay is shown in FIG. 2. 400 pmol/500 μl probescomplementary to those used in the labelling reaction were spotted onGene screen Plus nylon membranes (NEN), and crosslinked for 15 min witha UV transilluminator (Model TL33, UVP Inc., San Gabriel, Calif.). Themembranes were prehybridized in 0.5 M Na₂HPO₄ pH 7.2 and 1% SDS for 2hours. The labelled probes were added to 300 μl of 1×SSC and 6% PEG 1500heated to 80° C. for 5 min. The hybridisation was done over-night atroom temperature with agitation in a Cross Blot Dot Blot hybridisationchamber (Sebia, Moulinaux, France). The membrane was subsequently rinsedin 1×SSC, 1% SDS for 5 min, then 5 min in 0.1×SSC, 0.1% SDS, and finally5 min in 0.1 M Tris-HCl pH 7.5 and 0.15 M NaCl (antibody buffer). Atthis point the fluorescence was detected directly using a Typhoonscanner (Amersham-Pharmacia). The membranes were then blocked in for 1hour in blocking buffer: antibody buffer containing 1% skimmed milk(Difco, Detroit, Mich.). Blocking buffer containing 1/500antifluorescein HRP-conjugate was then added, and the hybridisationcontinued at room temperature for 1 hour. Finally, the membranes wererinsed for 30 min in antibody buffer, and the signals detected with 4 CNPlus chromogenic substrate according to the manufacturersrecommendations (NEN).

Quantification of scanned signals was carried out using the Imagemaster™Array software version 2.0 program and calculations were done withMicrosoft Excel 97 SR-2. TABLE 1 Primers and probes used in the PCRreactions Primer/ probe name Sequence (5′-3′) Head- 5′- TGC TAT GCG CGAGCT GCG - 3′ sequence Mon 810 Head Forward Mon810F1101MH AAT AAA GTG ACAGAT AGC TGG GCA primer Reverse Mon810R1101MH CCT TCA TAA CCT TCG CCC GBiotin Mon810 HH labelled TTT TTA CGA AGG ACT CTA ACG TTT AAC ATC probeCTT TGC CAT TTT T Mud F Mon810 Mud1101 ACG AAG GAC TCT AAC GTT TAA CATCCT TTG C Mud R Mon810 MudCap GCA AAG GAT GTT AAA CGT TAG AGT CCT TCG TBt11 Head Forward AHJ-2MH CGC ACA ATC CCA CTA TCC TT primer Reverse Bt11RDMH GCC TCC CAG AAG TAG ACG TC Biotin Bt11 HH TTT TTA AGA AAC CCT TACTCT AGC GAA GAT labelled CCT CTT TTT T probe Mud F Bt11 MudF AAG AAA CCCTTA CTC TAG CGA AGA TCC T Mud R Bt11 MudR AGG ATC TTC GCT AGA GTA AGGGTT TCT T Bt176 Head Forward Cry2 FMH CCC ATC GAC ATC AGC CTG AGC primerPepC-20MH ATC TCG CTT CCG TGC TTA GC Reverse Cry2 RMH CAG GAA GGC GTCCCA CTG GC Cry04 (SMT- GGT CAG GCT CAG GCT GAT GT CT96) MH Biotin Bt SynHH TTT TTA TGT CCA CCA GGC CCA GCA CGT TTT T labelled probe Mud F Bt176MudF BtSyn2 TCC ACC AGG CCC AGC ACG AAG BtSyn3 AGG CCC AGC ACG AAG CCG GBt176-cryA1-1904 TGA GCA ACC CCG AGG TGG AGG TG MudR Bt176 MudR BtSyn2MudR CCG GCT TCG TGC TGG GCC TGG TGG A Bt176 MudR2504 CAC CTC CAC CTCGGG GTT GCT CA 35 S Head Forward 35SH-1 GCT CCT ACA AAT GCC ATC A primer35SMH-F2 GAA GAT AGT GGA AAA GGA AGG TGG C 35SMH-F3 GGA AAC CTC CTC GGATTC CAT Reverse 35SMH-R1 CCC TTA CGT CAG TGG AGA TAT CAC AT 35SMHH-R2CTT GCT TTG AAG ACG TGG TTG G 35SMH-R3 GAT GCT CCT CGT GGG TGG G Mud F35S MudF GAA AGG CCA TCG TTG AAG ATG C 35S Mud2F TGC CGA CAG TGG TCC CAAAGA TGG A Mud R 35S MudR GGC ATC TTC AAC GAT GGC CTT TC 35S Mud2R TCCATC TTT GGG ACC ACT GTC GGC A Amp Head Forward Ampres FMH TGC TCA CCCAGA AAC GCT G primer Reverse Ampres RMH TTC TTC GGG GCG AAA ACT CTC MudF Amp pro GTA AAA GAT GCT GAA GAT CAG TTG GGT GCA Mud R Amp MudR TGC ACCCAA CTG ATC TTC AGC ATC TTT TAC Nos Head Forward Nos FMH GAA TCC TGT TGCCGG TCT TG primer Reverse Nos RMH AAT TTA TCC TAG TTT GCG CGC TA Mud FNos pro TTT ATG AGA TGG GTT TTT ATG ATT AGA GTC CCG Mud R Nos MudR CGGGAC TCT AAT CAT AAA AAC CCA TCT CAT AAA IPC Synthetic IPC CGC AGC GTTTCA AGC AGC ACA TCA TCG ATC TAA sequence TCG AGC AGA CGG TAC GAT CAG ACGCTG TCA TAC GCA TAA TCG ATA CGC GAT ACT GCC CGC TAA CTG G Forward IPC-FCGC AGC GTT TCA AGC AGC Reverse IPC-R CCA GTT AGC GGG CAG TAT CG HeadForward IPC-FMH CGC AGC GTT TCA AGC AGC primer Reverse IPC-RMH CCA GTTAGC GGG CAG TAT CG Mud F IPC pro AGC AGA CGG TAC GAT CAG ACG CTG T Mud RIPC MudR ACA GCG TCT GAT CGT ACC GTC TGC T Maize Ref gene Head ForwardZM1 FMH TTG GAC TAG AAA TCT CGT GCT GA primer Reverse ZM1 RMH GCT ACATAG GGA CCC TTG TCC T Biotin ZM1 HE TTT TTC AAT CCA CAC AAA CGC ACG CGTATT TTT labelled probe Mud F MudF CAA TCC ACA CAA ACG CAC C Mud R Mud RCGT GCG TTT GTG TGG ATT G* All Head primers contain the head sequence at the 5′- end in additionto the sequences listed

Example 1 Qualitative Multiplex Detection

This example shows that qualitative multiplex detection is possible. Themultiplex method was used to detect Bt11 corn (DNA from 2% referencematerial), Bt176 corn (1%) and Mon810 corn (1%) alone or in combinations(FIG. 3). A corn reference gene detection system was also included todetect corn DNA as such. Each sample was analysed with 2 parallels.

Example 2 Quantitative Nature of the PCR Assay

This example shows the quantitative nature of the PCR. Bt11 DNA wasdiluted with non-GM corn DNA to give different GM concentrations. Thesewere analysed using the multiplex assay (FIG. 4). The signals could bedetected directly by fluorescence scanning (not shown) or afterenzymatic enhancement (FIG. 4). The gradually fading signals as theconcentration of GMO decreases show that the assay is quantitative.

Example 3 Eight Plex Detection of GM Maize Constructs

1% Bt176 and 2% Bt11 DNA was detected in an eight-plex reaction (FIG.5). For Bt176 we obtained signals from the Bt176 construct specifictarget, the amp target, 35S promoter and maize specific reference geneand finally from the IPC control. The Bt11 sample gave signals with theBt11 construct specific PCR, the NOS terminator, the 35S promoter andthe maize reference gene in addition to the IPC. Weak signals were (andare essentially always) obtained with the amp primers even when no ampresistance genes from GMOs are present. This is probably due tocontamination with amp resistance gene from the DNA polymerasepreparation.

Example 4 Quantitative Nature of the 8-Plex PCR and the effect ofRemoving the “Head Primers” (Bipartite Primers) After the 1. PCR Step.

This experiment was done to show that it is necessary to remove the“headprimers” after the 1. PCR step to maintain the quantitative natureof the assay. Quantitative 8-plex PCR for detection of GMP corn wascarried out. Bt176 DNA and Bt11 DNA were kept constant at 0.7% in allsamples. Concentrations of Mon810 DNA was varied from 2.0 to 0%. In FIG.6A, the PCR was carried out in two steps: 1. PCR (10 cycles) usingspecific primers with a common “head” sequence. Primers were thendigested and the 2. PCR (30 cycles) is carried out using the common headprimer. FIG. 6B shows the same as FIG. 6A except that the specificprimers were not degraded before the 2.PCR step. FIG. 6A shows clearlythe quantitative nature of the assay as the Mon810 DNA signal isgradually fading as the concentration decreases. Even though the Mon810signals are decreasing in FIG. 6B, it is easily seen that the overallresults are dramatically influenced by not removing the “head primers”after the 1 PCR step. The relative signal strength from the differentPCRs is changed and the signals are generally weaker. This is mostprobably caused by different amplification efficiencies of the specificprimers with the headsequence and formation of primer dimers.

Example 5 Effect of Omitting the 2. PCR Step.

This example showed the effect of omitting the 2.PCR step (FIG. 7). Theexperiment was the same as in Example 4 (FIG. 6A) except that the 1. PCRstep using specific primers with head sequence was extended to 40 cyclesand the 2. PCR step was omitted. As in example 4, FIG. 6B performing thePCR with the headprimers present leads to different amplificationefficiencies for the different PCRs and some fragments (e.g. Bt176 andBt11) were not amplified.

Example 6 The Effect of Diluting the Template DNA

A reference mixture of 0.7% Bt176, 0.7% Bt11 and 0.7% Mon810 atdifferent dilutions was used as templates in the PCR (FIG. 8). We seethat the signals gradually fade as the template DNA is diluted, but thatthe dilution effect is relatively small down to 16 fold dilution.

Example 7 Quantitative Detection of Mon810 Alone and Together with Bt11

A dilution series containing different amounts of Mon810 DNA wasanalysed alone and in combination with 1% Bt11 in the samples. Thefluorescence signals after hybridisation of the labelled probes and theblot after HRP colouring are shown in FIG. 9. The fading of the Mon810signals as the amount of Mon810 DNA is lowered is clearly visible. The35S signal decreases in A down to zero as expected and down to a fixedlevel caused by the presence of Bt11 DNA in B. The other signals remainconstant. The fluorescence signals from Mon810 (FIG. 9 panel I) werequantified and plotted against the given concentrations (FIG. 10). Alinear response was observed up to 5% Mon810. Little difference wasobserved between parallels. The signal strengths remained the samewhether Bt11 DNA was present or not.

Example 8 Quantitative Detection of Mon810 Alone and Together with Bt11(Repetition)

To investigate the repeatability of the system, the experiment inexample 7 was repeated. A dilution series containing different amountsof Mon810 DNA was analysed alone and in combination with 1% Bt11 in thesamples. The fluorescence signals and the blot after HRP colouring areshown in FIG. 11. The fading of the Mon810 signals as the amount ofMon810 DNA is lowered is again clearly visible, although the signalshave reached some degree of saturation and the difference betweensignals at higher concentrations of Mon810 is smaller. The 35S signaldecreases in A down to zero as expected and down to a fixed level causedby the presence of Bt11 DNA in B. The other signals remain constant. Thefluorescence signals from Mon810 were again quantified and plottedagainst the given concentrations (FIG. 12). An almost linear responsewas observed up to 2% Mon810. The signal at 5% Mon810 was lower thanexpected probably due to saturation of the probe (all probe moleculeswere already labelled). The difference between parallels were greaterthan at example 7. Again the signal strengths remained the same whetherBt11 DNA was present or not.

Example 9 Quantitative Detection of Bt176 Alone and Together with Mon810

The experiment was performed as in example 6, except that the amount ofBt176 was varied and Mon810 was kept constant. A dilution seriescontaining different amounts of Bt176 was analysed alone and incombination with 1% Mon810 in the samples. The flueorescence signals andthe blot after HRP colouring are shown in FIG. 13. The fading of theBt176 signals as the amount of Bt176 DNA is lowered is clearly visible.The 35S signal and the amp signal decrease in A down to zero asexpected. 35S decreases down to a fixed level caused by the presence ofMon810 DNA in B. The other signals remain constant. The fluorescencesignals from Mon810 were again quantified and plotted against the givenconcentrations (FIG. 14). Also here a (close to) linear response wasobserved.

Table 2 below gives details of primers and probes for use in Examples10, 11 and 12 as well as preferred oligonucleotides for use in theearlier Examples. In this table there are no biotin labelled probes andas shown in Table 1, it is understood that a probe complementary to e.g.Bt11 MudF may also be used in the labelling and capture step. TABLE 2Primers, probes and template DNA used in multiplex-PCR and 5′-nucleasePCR. Template Orientation Name Sequence (5′->3′) Primers and probes forthe multiplex system HEAD H TGC TAT GCG CGA GCT GCG sequence Mon810¹Sense^(§) Mon810F1101MH H-AAT AAA GTG ACA GAT AGC TGG GCA Antisense^(§)Mon810R1101MH H-CCT TCA TAA CCT TCG CCC G Probe^(**) Mon810 Mud1101 ACGAAG GAC TCT AAC GTT TAA CAT CCT TTG C Bt11 Sense AHJ-2MH H-CGC ACA ATCCCA CTA TCC TT Antisense Bt11 RBMH H-GCC TCC CAG AAG TAG ACG TC ProbeBt11 MudF AAG AAA CCC TTA CTC TAG CGA AGA TCC T Bt176 Sense PepC-20MHH-ATC TCG CTT CCG TGC TTA GC Antisense Cry04(SMT-CT96)MH H-GGT CAG GCTCAG GCT GAT GT Probe Bt176-cryA1-1904 TGA GCA ACC CCG AGG TGG AGG TGT25² Sense T25 FMHB H-CCA GTT AGG CCA GTT ACC CAG A Antisense T25 RMHBH-TGG GAA CTA CTC ACA CAT TAT TAT AGA GAG Probe T25 Mud AGA CTG GTG ATTTCA GCG GGC ATG GA21² Sense GA21 FMHB H-AGC CTC GGC AAC GTC AGCAntisense GA21 RMHB H-TCT CCT TGA TGG GCT GCA G Probe GA21 MUDR AAG GATCCG GTG CAT GGC CGG Probe capture GA21 MUD GCC GGC CAT GCA CCG GAT CCT TDBT418 Sense DBT418 FMHB H-GTC ATT TCA GGA CCA GGA TTC AC AntisenseDBT418 RMHB H-CCT CTA TTC TGG ATG TTG TTG CC Probe DBT418 MUDR GAA GAATTC AGC CTA ACC AAG TCG CCT C CBH351 Sense CBH351 FMHB H-GGT CAG ATC GTGAGC TTC TAC CA Antisense CBH351 RMHB H-CGC ATG AAA GCT TCC CAG AT ProbeCBH351 MUD GCT GAA CAC CCT GTG GCC AGT GAA 35S Sense 35SH-1 H-GCT CCTACA AAT GCC ATC A Antisense 35SMHH-R2 H-CTT GCT TTG AAG ACG TGG TTG GProbe 35S Mud2F TGC CGA CAG TGG TCC CAA AGA TGG A Amp Sense Ampres FMHH-TGC TCA CCC AGA AAC GCT G Antisense Ampres RMH H-TTC TTC GGG GCG AAAACT CTC Probe Amp pro GTA AAA GAT GCT GAA GAT CAG TTG GGT GCA Nos SenseNos FMH H-GAA TCC TGT TGC CGG TCT TG Antisense Nos RMH H-AAT TTA TCC TAGTTT GCG CGC TA Probe Nos pro TTT ATG AGA TGG GTT TTT ATG ATT AGA GTC CCGIPC Forward IPC-FMH H-CGC AGC GTT TCA AGC AGC Reverse IPC-RMH H-CCA GTTAGC GGG CAG TAT CG Probe IPC pro AGC AGA CGG TAC GAT CAG ACG CTG T ZMref Sense ZM1 FMH H-TTG GAC TAG AAA TCT CGT GCT GA Antisense ZM1 RMHH-GCT ACA TAG GGA GCC TTG TCC T Probe MudF CAA TCC ACA CAA ACG CAC GPrimers and DNA used for template construction T25 T25 1-5′ GCC AGT TAGGCC AGT TAC CCA T25 1-3′ TGA GCG AAA CCC TAT AAG AAC CCT GA21 GA21 F AGCCTC GGC AAC GTC AGC GA21 R TGT CCT TGA TGG GCT GCA G DBT418 TemplateDBT418hele GTC ATT TCA GGA CCA GGA TTC ACT GGA GGC GAC TTG GTT AGG CTGAAT TCT TCC GGC AAC AAC ATC CAG AAT AGA GG CBH351 Template CBH351heleGGT CAG ATC GTG AGC TTC TAC CAG TTC CTG CTG AAC ACC CTG TGG CCA GTG AACGAC ACC GCC ATC TGG GAA GCT TTC ATG CG 35S Sense P35S 1-5′ ATT GAT GTGATA TCT CCA CTG ACG T Antisense T35S 1-3′ ACT AAG GGT TTC TTA TAT GCTCAA CA IPC Forward IPC-F CGC AGC GTT TCA AGC AGC Reverse IPC-R CCA GTTAGC GGG CAG TAT CG Template IPC-T CGC AGC GTT TCA AGC AGC ACA TCA TCGATC TAA TCG AGC AGA CGG TAC GAT CAG ACG CTG TCA TAC GCA TAA TCG ATA CGCGAT ACT GCC CGC TAA CTG G 5′-nuclease PCR primers and probes^(§§) Bt11Sense Fbt11-enhpatjun-AHJ-1 CTT GGC GGC TTA TCT GTC TC AntisenseRbt11-enhpatjun-AHJ-2 GCT GCT GTA GCT GGC CTA AT ProbeFam-Bt11-enh-pat^(*) TCG ACA TGT CTC CGG AGA GGA GAC C Bt176 ProbeBt176-CryA1t CTG AGC AAC CCC GAG GTG GAG GT T25 Sense T25 1-5′ GCC AGTTAG GCC AGT TAC CCA Antisense T25 1-3′ TGA GCG AAA CCC TAT AAG AAC CCTProbe T25 pro^(*) GCA TGC CCG CTG AAA TCA CCA GTC T DBT418 Sense DBT418F GTC ATT TCA GGA CCA GGA TTC AC Antisense DBT418 R CCT CTA TTC TGG ATGTTG TTG CG Probe DBT418 pro^(*) GGA GGC GAC TTG GTT AGG CTG AAT TCT TCCBH351 Probe CBH351 pro^(*) TGC TGA ACA CCC TGT GGC CAG TGA Zein³ SenseZetm1 TGT TAG GCG TCA TGA TCT GTG G Antisense Zetm3 TGC AGC AAC TGT TGGCCT TAC Probe Zetmp^(*) ATC ATC ACT GGC ATC GTC TGA AGC GG^(§)All sense and antisense primers used in the MQDA-PCR contain theHEAD sequence, designated by an H at the 5′-end in addition to the givensequence.^(*)All 5′-nuclease PCR probes contain 5′FAM (6-FAM) and 3′Tamra.^(§§)All filter bound capture probes are complementary to theircorresponding probes## Only primers and probes different from those used in the MQDA-PCR arelisted. All 5′-nuclease primers are without the HEAD sequence.¹Holck, A., M. Va+E,umla itilingom, L. Didierjean, and K. Rudi. 2002.5′-nuclease PCR for quantitative event-specific detection of thegenetically modified Mon810 MaisGard maize. European Food Research andTechnology 214: 449-453.²Matsuoka, T., Kuribara, H., Akiyama, H., Miura, H., Goda, Y., Kusakabe,Y., Isshiki, K., Toyoda, M., Hino,A. 2001. A multiplex PCR method ofdetecting recombinant DNAs from five lines of genetically modifiedmaize. Journal of the Food Hygienic Society of Japan 42: 24-32.³Vaïtilingom, M., Pijnenburg, H., Gendre, F., Brignon, P. 1999.Real-Time Quantitative PCR detection of genetically modified MaximizerMaize and Roundup Ready Soybean in some representative foods. Journal ofagricultural and food chemistry 47:5261-5266

Example 10 Twelve-Plex PCR to Detect Seven Different GM Maize

The multiplex system was expanded from an eight-plex PCR to atwelve-plex PCR through the inclusion of primers for detection of themaize constructs CBH351, DBT418, GA21 and T25 (FIG. 15). Mixturescontaining 0.7 or 1.0% of each of all seven different GM constructs wereamplified in one reaction together with the amplicons from amp, nos,35S, IPC and the maize reference genes. When CBH351, DBT418, GA21 andT25 were amplified separately, a dose response was observed (FIG. 16).No cross reactions during hybridisation were detected. The specificsignals from the mixtures of the seven GM constructs were generallyweaker than those obtained with pure samples (FIG. 16). This may comefrom a slightly higher frequency of side reactions when multiple targetsare amplified simultaneously. In some cases the standard curves deviatefrom linearity. This is particularly observed when signals are strongand stems from saturation of targets during the probe labeling reaction.This may be adjusted by lowering the number of labeling cycles. Sincereference materials are used in each experiment quantifications arenevertheless possible.

Example 11 Analysis of 17 Food and Feed Samples from USA

Seventeen different food and feed samples were screened using thetwelve-plex system described in Example 10 (FIGS. 15 and 16). Tensamples were GM positive, and seven GM negative (GM content <0.1%).Eight GM positive samples contained Mon 810, eight contained Bt11 andseven contained Bt176. One sample contained appreciable amounts of GA21,a few more samples harboured small mounts of GA21 and in one samplesmall amounts of T25 was observed. No maize material containing theconstructs CBH 351 or DBT418 was detected. These results are reasonablesince the latter four GM maizes are either withdrawn from the market orknown to be not very widespread. All the GM negative samples also testednegative when analysed for Bt11, Bt176 and Mon810 by 5′-nuclease PCR.Two GM positive samples contained only marginal amounts of DNA as judgedby the increase in intensity of the IPC signal and the Ct values of the5′ nuclease PCR. Quantification of GM content in these samples wastherefore not possible (Ct for GM constructs >40).

Example 12 Comparison of Quantification Between Eightplex-PCR and 5′Nuclease PCR (Taqman PCR)

Seven GM positive samples and one negative sample were selected for acomparative quantitative study between the eight-plex PCR and5′-nuclease PCR for the constructs Mon810, Bt11 and Bt176 (FIGS. 18 and19). The samples were analysed together with known reference samples.The results of the quantifications are summarized in Table 3 below. Boththe eightplex-PCR and 5′ nuclease PCR require known standards forquantification. That is, accurate quantifications can only be obtainedif reference materials are analysed in the same experiment. Thequantitative results obtained using the calibration curves based on thereference material gave good agreement between the eightplex-PCR and the5′-nuclease approach. The multiplex method accurately identified sampleswith high and low content of GM material. In 20 out of 23 analyses thesamples could be quantified as containing more than 2%, between 1 and2%, between 1% and 0.1% or less than 0.1% by both methods. If all theother GM negative samples (<0.1% GM material) are included thecorresponding figures are 43 out of 47, respectively. The averagestandard deviations for the multiplex PCR is comparable to those of the5′ nuclease PCRs (FIG. 20). When pooled, the pooled average standarddeviation was statistically not different from that of the pooledstandard deviation of 5′ nuclease PCR. The pooled average standarddeviation of the multiplex PCR was also not different from the pooledstandard deviation of a 5′ nuclease PCR ringtrial encompassing six maizemeal samples analysed by nine laboratories in Europe. TABLE 3 GMquantification comparisons between eight-PCR and 5′-nuclease PCR on foodand feed samples Mon 810 Bt 11 Bt 176 Sample # Origin 8plex-PCR 5′nuclease 8plex-PCR 5′ nuclease 8plex-PCR 5′ nuclease 1 Maize grain^($)1.1 ± 0.2 1.1 ± 0.3 <0.1 <0.1 Nd* 18.6 ± 9.1  2 Dog food >2 4.4 ± 3.5 >211.5 ± 5.2  0.8 ± 0.2 0.6 ± 0.1 3 Dog food >2 8.5 ± 2.4 >2 5.2 ± 0.0 0.8± 0.3 0.5 ± 0.1 5 Chicken feed >2 35.1 ± 23   0.6 ± 0.4 2.0 ± 0.9 0.7 ±0.0 2.1 ± 0.2 9 Instant corn mix <0.1 <0.1 0.8 ± 0.5 0.2 ± 0.1 <0.1 <0.16 Corn meal 0.3 ± 0.0 0.2 ± 0.2 0.2 ± 0.3 0.1 ± 0.0 1.6 ± 0.8 1.5 ± 0.3 7** Whole kernel corn <0.1 <0.1 <0.1 <0.1 <0.1 <0.1 8 Yellow cornmeal >2 13.6 ± 2.0  1.4 0.6 ± 0.7 >2 12.9 ± 2.7 ^($)horse feed*nd not determined**A negative sample included

Example 13

Use of Uracil DNA Glycosylase (UNG) to Degrade modified bipartiteprimers

Bipartite primers were designed to amplify the glnA gene fromCampylobacter jejuni and constructed to contain uracil instead ofthymine at all (appropriate) positions. The “head” primers consisted ofpart B only and did not contain any uracil. Forward;5′GCAGGCUGCUCAUGUCUG UAGGAACUUGGCAUCAUAUUACC3′ Reverse;5′GCAGGCUGCUCAUGUCUG UUGGACGAGCUUCUACUGGC3′ Head; 5′GCAGGCTGCTCATGTCTG3′

Both sets of primers were added at the start of the PCR reaction. Afirst amplification reaction was performed and after 10 amplificationcycles UNG was added to degrade the bipartite primers. After thisdegradation step, amplification of the target sequence was stillobserved.

Example 14 Use of Uracil DNA Glycosylase (UNG) to Generate “HeadPrimers” from the Bipartite Primers

Bipartite primers were designed to amplify the glnA gene fromCampylobacter jejuni. Part A of these primers was specific for the gluAsequence and contained uracil instead of thymine, whereas part B did notcontain any uracil. Forward; 5′GCAGGCTGCTCATGTCTGUAGGAACUUGGCAUCAUAUUACC3′ Reverse; 5′GCAGGCTGCTCATGTCTGUUGGACGAGCUUCUACUGGC3′

At the start of the PCR reaction, the bipartite primers were the onlyprimers present. After 5 amplification cycles UNG was added to degradepart A of the bipartite primers. This degradation was detected by gelelectrophoretic analysis. This reaction thus generated primers whichonly contained part B, and could participate in a second amplificationreaction. Amplification was indeed observed after the UNG degradationstep.

1. A method of simultaneously amplifying a plurality of target sequenceswithin sample nucleic acid which comprises: (a) contacting said samplenucleic acid with one or more primer pairs under conditions which allowhybridisation of the primers to the sample nucleic acid, each primerhaving a bipartite structure A-B wherein part A is specific for aparticular target sequence within the sample nucleic acid and part B isa constant sequence which is common to all primers or is common amongstall forward primers with a different sequence common amongst all reverseprimers; (b) performing a first amplification reaction; (c) degradingthe bipartite primers or separating them from the amplification productsof the first amplification reaction; (d) contacting the amplificationproducts from the first amplification reaction with primers whichcomprise part B of the bipartite primers or a nucleotide sequence whichis substantially identical to part B, under conditions which allowhybridisation of the primers to the amplification products; and (e)performing a second amplification reaction.
 2. A method as claimed inclaim 1 wherein the constant region B of the bipartite primers is commonbetween both forward and reverse primers.
 3. A method as claimed inclaim 1 wherein the constant region B is 10-40 nucleotides in length. 4.A method as claimed in claim 1 wherein the first amplification reactioncomprises no more than 25 amplification cycles.
 5. A method as claimedin claim 1 wherein step (c) comprises contacting the bipartite primerswith a DNA-modifying enzyme so as to cause degradation thereof.
 6. Amethod as claimed in claim 5 wherein step (c) comprises contacting thebipartite primers with an exonuclease so as to cause degradationthereof.
 7. A method as claimed in claim 5 wherein the bipartite primerscontain one or more uracil residues.
 8. A method as claimed in claim 7wherein the modifying enzyme is uracil DNA glycosylase.
 9. A method asclaimed in claim 7 wherein the bipartite primers contain no thymineresidues.
 10. A method as claimed in claim 7 wherein the bipartiteprimers contain uracil in part A but not part B.
 11. A method as claimedin claim 1 wherein step (c) comprises isolating the amplificationproducts from the initial reaction mixture.
 12. A method as claimed inclaim 11 wherein the amplification products of the first amplificationreaction are captured on a solid support.
 13. A method as claimed inclaim 12 wherein the amplification products are contacted with a probeincorporating a binding partner for a binding moiety provided on saidsolid support.
 14. A method as claimed in claim 1 wherein all of steps(a)-(e) are performed in one reaction vessel.
 15. A method as claimed inclaim 1 wherein 4 or more target sequences are amplified simultaneously.16. A method as claimed in claim 1 wherein one or more of the targetsequence comprises a non-naturally occurring nucleotide sequence.
 17. Amethod as claimed in claim 16 wherein the target sequence comprisesregions which are not naturally found in juxtaposition.
 18. A method asclaimed in claim 1 wherein one or more of the primer pairs is designedto hybridise either side of a junction region between a regulatoryregion and a coding region within sample nucleic acid.
 19. A method asclaimed in claim 1 wherein the sample nucleic acid comprises hostorganism nucleic acid and a genetically engineered construct.
 20. Amethod as claimed in claim 19 wherein one or more of the targetsequences spans a region which comprises both host organism nucleic acidand inserted nucleic acid from the genetically engineered construct. 21.A method as claimed in claim 1 wherein the products of the secondamplification reaction are contacted with a plurality of differentprobes designed to hybridise to the target sequences under conditionswhich allow hybridisation thereof.
 22. A method as claimed in claim 21wherein the probes which hybridise to the target sequences are labelledat their 3′ end.
 23. A method as claimed in claim 22 wherein thelabelled probes are captured on a solid support.
 24. A method as claimedin claim 1 wherein a known concentration of a control nucleic acidsequence is added to the sample nucleic acid prior to the firstamplification reaction.
 25. A method as claimed in claim 20 wherein ahost species specific sequence is co-amplified with said target sequencewhich spans a region which comprises both host organism nucleic acid andinserted nucleic acid from the genetically engineered construct.
 26. Akit for use in a method of nucleic acid amplification which comprises:(a) a plurality of bipartite primer pairs of form A-B as defined inclaim 1; (b) means for degrading the bipartite primers or for separatingthem from the amplification products of a first amplification reaction;and optionally (c) primers which comprise part B of the bipartiteprimers of component (a) or a nucleotide sequence which is substantiallyidentical to part B of said primers.